The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88−/−, Trif−/−, MyD88−/−Trif−/−, MK2−/−, and Zfp36−/− mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)–induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.

中文翻译：

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Tristetraprolin调节鼠巨噬细胞在补药Toll样受体4（TLR4）信号传导期间的坏死病

坏死体是细胞信号传导所必需的蛋白质复合物，其导致坏死性坏死，这也取决于肿瘤坏死因子受体（TNF-R）信号传导。TNFα促进坏死性坏死，其表达受促分裂原激活蛋白（MAP）激酶激活的蛋白激酶2（MK2）促进，但受RNA结合蛋白tristetraprolin（TTP，由Zfp36基因编码）抑制。我们已经从WT，MyD88-/-，Trif-/-，MyD88-/-Trif-/-，MK2-/-和Zfp36-/-小鼠中用分级剂量的脂多糖（LPS）和各种抑制剂刺激了小鼠巨噬细胞评估各种基因在Toll样受体4（TLR4）引起的尸检中的作用。分别通过免疫印迹，ELISA和细胞死亡试验评估了坏死信号传导，细胞因子产生和细胞死亡。我们观察到在TLR4信号转导期间，坏死体激活是通过衔接子蛋白MyD88和TRIF介导的，这被MK2抑制。在缺乏MK2介导的坏死体激活的情况下，脂多糖诱导的TNFα表达急剧降低，但是即使在低TNFα水平下，缺乏MK2的细胞也对坏死病高度敏感。相反，在补剂TLR4信号转导期间，即使禁用MK2，WT细胞也不会发生坏死性坏死。值得注意的是，尸检仅在没有TTP的情况下发生，并由TNFα的表达和JUN N-末端激酶（JNK）的激活介导。这些结果表明，TTP在抑制TNFα/ JNK诱导的坏死因子信号传导和由此产生的细胞毒性中起重要作用。脂多糖诱导的TNFα表达急剧降低，但是即使在低TNFα水平下，缺乏MK2的细胞也对坏死高度敏感。相反，在补剂TLR4信号转导期间，即使禁用MK2，WT细胞也不会发生坏死性坏死。值得注意的是，尸检仅在没有TTP的情况下发生，并由TNFα的表达和JUN N端激酶（JNK）的激活介导。这些结果表明，TTP在抑制TNFα/ JNK诱导的坏死因子信号传导和由此产生的细胞毒性中起重要作用。脂多糖诱导的TNFα表达急剧降低，但是即使在低TNFα水平下，缺乏MK2的细胞也对坏死高度敏感。相反，在补剂TLR4信号转导期间，即使禁用MK2，WT细胞也不会发生坏死性坏死。值得注意的是，尸检仅在没有TTP的情况下发生，并由TNFα的表达和JUN N-末端激酶（JNK）的激活介导。这些结果表明，TTP在抑制TNFα/ JNK诱导的坏死因子信号传导和由此产生的细胞毒性中起重要作用。坏死仅在不存在TTP的情况下发生，并由TNFα的表达和JUN N-末端激酶（JNK）的激活介导。这些结果表明，TTP在抑制TNFα/ JNK诱导的坏死因子信号传导和由此产生的细胞毒性中起重要作用。坏死仅在不存在TTP的情况下发生，并由TNFα的表达和JUN N末端激酶（JNK）的激活介导。这些结果表明，TTP在抑制TNFα/ JNK诱导的坏死因子信号传导和由此产生的细胞毒性中起重要作用。